PROJECT SUMMARY Coronaviruses are a diverse family of viruses infecting many animals including humans. The 21st century is now experiencing its third outbreak of a novel pathogenic coronavirus that has crossed from an animal host into humans for the first time. The outbreak of SARS-CoV-2 is unprecedented among human coronaviruses in its size and the speed of its spread. Countering this viral outbreak will require a detailed mechanistic understanding of virus protein function. The goals of this project are to gain highly detailed information about the SARS-CoV-2 replication complex using single-particle cryo-electron microscopy. We will use this high-resolution imaging technique to determine structures of the SARS-CoV-2 polymerase complex bound to substrates and small molecule antiviral drugs. We will complement these structural analyses with a detailed biochemical study of protein-protein and protein-RNA interactions and the influence of these interactions on SARS-CoV-2 polymerase activity. These studies include mutagenesis of key interfaces in the protein complex and testing of the recombinant proteins in polymerase activity assays. These studies have the potential to illuminate the mechanisms used by the SARS-CoV-2 polymerase complex to replicate its viral genome and provide a mechanistic understanding of antiviral therapeutic action key to the development of novel antiviral therapeutics to treat COVID-19.